Several kinds of potential nucleic acid therapeutics have been explored over the last two decades, including RNA inhibitors such as antisense, ribozymes (catalytic RNAs), and artificial ligand inhibitors (“aptamers”). These therapeutics are designed to silence gene expression, and thus to alleviate the effects of undesirable genes, be they endogenous to an organism or exogenous, such as bacterial or viral in origin. Because it is difficult to apply these to cells externally, there has been significant interest in expressing them within cells. However, expression of these therapeutics intracellularly has proved quite difficult as well; this difficulty is thought to be due to several factors. These include, for RNA-based therapeutics as an example, the considerations of finding their targets, folding into the effective configuration, and possibly interacting with the appropriate proteins while avoiding interactions with inappropriate proteins. There have been isolated promising results (see, for example, Bertrand, E. et al., RNA3: 75-88 (1997); Good, P D et al. Gene Therapy 4:45-54 (1997)), but no therapeutics have yet resulted.
RNA Interference
RNA interference, or RNAi, is an endogenous, efficient, and potent gene-specific silencing technique that uses double-stranded RNAs (dsRNA) to mark a particular transcript for degradation in vivo. First discovered in the nematode Caenorhabditis elegans, it has since been found to operate in a wide variety of organisms. RNAi is believed to be effected by dsRNAs ˜21-25 nucleotides long, called short interfering RNAs (siRNAs), which are endogenously produced through the degradation of long dsRNA molecules by an RNAse III-related nuclease called Dicer. Once formed, the siRNAs associate with a multiprotein complex called RISC (RNA-Induced Silencing Complex), which targets the homologous RNA by Watson-Crick base pairing for sequence specific degradation of mRNA.
This sequence-specific degradation of mRNA results in knocking down (partially or completely) the targeted gene. Thus RNAi provides an alternative to presently available methods of knocking down (or out) a gene or genes. This method of knocking down gene expression can be used therapeutically or for research (e.g., to generate models of disease states, to examine the function of a gene, to assess whether an agent acts on a gene, or to validate targets for drug discovery).
There are two main approaches to employing RNAi in cells. In the first approach, an expression construct (for either integrative or transient expression), which encodes an RNA including the desired RNAi sequences, is introduced into the target cells. The endogenous dicer enzyme recognizes and processes this RNA into the desired ˜21-23 nucleotide siRNAs, which then enter an effector complex, RISC. In the second approach, the siRNAs (in either single-stranded antisense or double-stranded form) are introduced directly into the cell and directly enter the RISC complex. In both of these approaches, guided by the antisense strand of the siRNA, the active form of RISC (activated by the ATP-dependent unwinding of the siRNA duplex) recognizes and suppresses gene expression through mRNA degradation or prevention of protein synthesis.
RNAi has been studied in a variety of systems. Fire et al., Nature, 391: 806 (1998), were the first to observe RNAi in C. elegans. Wianny and Goetz, Nature Cell Biol., 2:70 (1999), describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., Nature, 404:293 (2000), describe RNAi in Drosophila cells transfected with dsRNA. Elbashir at al., Nature, 411:494 (2001), describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells by including human duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including embryonic kidney and HeLa cells.
Recent work in Drosophila embryonic lysates (Elbashir et al., EMBO J., 20:6877(2001)) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete disubstitution of one or both siRNA strands with 2′deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., EMBO J., 20:6877 (2001)). Other studies have indicated that a 5′-phosphate on the target complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., Cell, 107:309 (2001)).
Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well-tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity. In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Application No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom; however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, it is unclear as to what extent these modifications would be tolerated in siRNA molecules.
The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et. al. International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, Chem. Biochem., 2:239-245 (2001), doubts that RNA can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for Introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the Identification of specific genes Involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA constructs for use in facilitating gene silencing in targeted organisms.
Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., Molecular Cell, 6:1977-1087 (2000), describe chemically modified siRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/38551, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT Publication No. WO 01/53745, describe certain methods for isolating a neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/6334, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain dsRNAs. Echeverri et al., International PCT Publication No. WO 02.38805, describe C. elegans genes identified via RNAi. Kruetzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describe certain methods for inhibiting gene expression using RNAi. Graham et al., International PCT Publication Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (greater than 25 nucleotide) constructs that mediate RNAi.
Delivering siRNAs directly to whole vertebrate animals is more problematic than it is for invertebrates or cell lines. Conventionally constructed oligonucleotides have poor serum stability, are susceptible to nuclease degradation, and cannot easily cross cell membranes. Two groups of scientists independently employed a “hydrodynamic transfection method” to deliver naked siRNAs to mice via tail-vein Injection. A. P. McCaffrey et al., “Gene expression: RNA interference in adult mice,” Nature, 418:38-9 (2002); D. J. Lewis et al., “Efficient delivery of siRNA for inhibition of gene expression in postnatal mice,” Nat. Genet., 32:107-8 (2002). While these scientists observed downregulation of a reporter gene by 80%-90% in the liver, kidney, spleen, lung, and pancreas, the effect was relatively short-lived, lasting only a few days.
Thus, there is a need to produce siRNAs that have improved characteristics for both in vitro delivery to cells and in particular, in vivo delivery for therapeutic applications.
The design of nucleic acids, particularly oligonucleotides, for in vivo delivery requires consideration of various factors including binding strength, target specificity, serum stability, resistance to nucleases and cellular uptake. A number of approaches have been proposed in order to produce oligonucleotides that have characteristics suitable for in vivo use, such as modified backbone chemistry, formulation in delivery vehicles and conjugation to various other moieties. Therapeutic oligonucleotides with characteristics suitable for systemic delivery would be particularly beneficial.
Oligonucleotides with modified chemical backbones are reviewed in Micklefield, Backbone modification of nucleic acids: synthesis, structure and therapeutic applications, Curr. Med. Chem., 8(10):1157-79, 2001 and Lyer et al., Modified oligonucleotides-synthesis, properties and applications, Curr. Opin. Mol. Ther., 1(3): 344-358, 1999.
Examples of modified backbone chemistries include:                peptide nucleic acids (PNAs) (see Nielsen, Methods Mol. Biol., 208:3-26, 2002),        locked nucleic acids (LNAs) (see Petersen & Wengel, Trends Biotechnol., 21(2):74-81, 2003),        phosphorothioates (see Eckstein, Antisense Nucleic Acid Drug Dev., 10(2):117-21, 2000),        methylphosphonates (see Thiviyanathan et al., Biochemistry, 41(3):827-38, 2002),        phosphoramidates (see Gryaznov, Biochem. Biophys. Acta, 1489(1):131-40, 1999; Pruzan et al., Nucleic Acids Res., 30(2):559-68, 2002), and        thiophosphoramidates (see Gryaznov et al., Nucleosides Nucleotides Nucleic Acids, 20(4-7):401-10, 2001; Herbert et al., Oncogene, 21 (4):638-42, 2002).        
Each of these types of oligonucleotides has reported advantages and disadvantages. For example, peptide nucleic acids (PNAs) display good nuclease resistance and binding strength, but have reduced cellular uptake in test cultures; phosphorothioates display good nuclease resistance and solubility, but are typically synthesized as P-chiral mixtures and display several sequence-non-specific biological effects; methylphosphonates display good nuclease resistance and cellular uptake, but are also typically synthesized as P-chiral mixtures and have reduced duplex stability. The N3′→P5′ phosphoramidate internucleoside linkages are reported to display favorable binding properties, nuclease resistance, and solubility (Gryaznov and Letsinger, Nucleic Acids Research, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995; Skorski et al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). However, they also show increased acid lability relative to the natural phosphodiester counterparts (Gryaznov et al., Nucleic Acids Research, 24:1508-1514, 1996). Acid stability of an oligonucleotide is an important quality given the desire to use oligonucleotide agents as oral therapeutics. The addition of a sulfur atom to the backbone in N3′→P5′ thiophosphoramidate oligonucleotides provides enhanced acid stability.
As with many other therapeutic compounds, the polyanionic nature of oligonucleotides reduces the ability of the compound to cross lipid membranes, limiting the efficiency of cellular uptake. Various solutions have been proposed for increasing the cellular uptake of therapeutic agents, including formulation in liposomes (for reviews, see Pedroso de Lima et al., Curr. Med. Chem., 10(14):1221-1231, 2003 and Miller, Curr. Med. Chem., 10(14):1195-211, 2003) and conjugation with a lipophilic moiety. Examples of the latter approach include: U.S. Pat. No. 5,411,947 (Method of converting a drug to an orally available form by covalently bonding a lipid to the drug); U.S. Pat. No. 6,448,392 (Lipid derivatives of antiviral nucleosides: liposomal incorporation and method of use); U.S. Pat. No. 5,420,330 (Lipo-phosphoramidites); U.S. Pat. No. 5,763,208 (Oligonucleotides and their analogs capable of passive cell membrane permeation); Gryaznov & Lloyd, Nucleic Acids Research, 21:5909-5915, 1993 (Cholesterol-conjugated oligonucleotides); U.S. Pat. No. 5,416,203 (Steroid modified oligonucleotides); WO 90/10448 (Covalent conjugates of lipid and oligonucleotide); Gerster et al., Analytical Biochemistry, 262:177-184 (1998) (Quantitative analysis of modified antisense oligonucleotides in biological fluids using cationic nanoparticles for solid-phase extraction); Bennett et al., Mol. Pharmacol., 41:1023-1033 (1992) (Cationic lipids enhance cellular uptake and activity of phophorothioate antisense oligonucleotides); Manoharan et al., Antisense and Nucleic Acid Drug Dev., 12:103-128 (2002) (Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery and mechanism of action); and Fiedler et al., Langenbeck's Arch. Surg., 383:269-275 (1998) (Growth inhibition of pancreatic tumor cells by modified antisense oligodeoxynucleotides).